Early work by Zimmerman (U.S. Pat. No. 4,292,408), Sowers (U.S. Pat. No. 4,622,302) and Yaoita et al. (1988), among others, established that the outer cell membrane could be altered by the presence of a transient electric field. Much of the original interest in this process related to cellular fusion, where two or more cells are joined. The enduring technology, however, has been that of electroporation, where temporary pores are created in the outer cell membrane by the application of an electric field to allow material from the surrounding medium to move into the cell.
The treatment of suspended cells in a cuvette that has aluminum electrodes built into opposing walls has become a widely used electroporation technique. Commercial electroporation systems for this process are available from a number of lab instrumentation suppliers such as BioRad, BTX, Invitrogen and Eppendorf.
Initially, adherent cells were electroporated using suspension cell equipment by first detaching the cells from the substrate on which they were growing. Because this can substantially disturb adherent cells, various techniques were developed for subjecting adherent cells to an electric pulse without detaching them from the substrate. The BTX Petri Pulser™ is a commercially available unit consisting of a set of parallel, thin, gold coated plates that rest on edge in a Petri dish where cells are growing while a pulse is delivered between the plates which alternate in polarity. However, many cells are damaged in this process and cleaning the elaborate electrode set complicates the process.
Recently, techniques employing the processes used in microelectronic silicon chip manufacture have been applied to make “lab on a chip” systems that use electroporation to treat small numbers of suspended or adherent cells. Khine et al. (2005) describes a polymeric chip that can selectively immobilize and locally electroporate single cells. Yu-Cheng Lin et al. (2001) describes an electroporation microchip consisting of a defined cell culture cavity region with thin-film electrodes made of titanium and gold, fabricated on a glass slide using micro-fabrication technologies, which include evaporation, photolithography and wet-etching methods.
Continuing work in the field of electroporation indicates the need for a method for introducing materials into cells that is not well met by existing alternative procedures. Micro-injection, where material is injected into cells through the cell wall using small stabilized needles, is very slow and requires expensive, sophisticated manipulators. Scrape loading, where a sharp blade slashes through a monolayer of adherent cells, can cause some cells to take in material through their wounded parts before healing. However, such harsh treatment of the cells raises concerns about the reliability of subsequent conclusions.
Calcium phosphate transfection predates electroporation and continues to be used though it is appropriate only for nucleic acids. Liposome transfection has also been used, though making appropriate liposomes containing the desired material and having them fuse with the cells complicates the process.
Over the past two decades, with advances in genetics research, the ability of electroporation to introduce genetic material into cells has been the application most in demand and it will continue to be very important. In recent years, research into the role of smaller molecules such as peptides, and the use of fluorescent markers for the study of cellular functions, are expanding the scope of applications involving electroporation, particularly with respect to adherent cells.
U.S. Pat. No. 5,232,856 to Firth describes a method for providing a uniform electric field over an area of cells growing on a conductive, transparent electrode made of indium tin oxide, by using a specific geometry of a second electrode in contact with the electroporation medium immediately above the cells. Boccaccio et al. (1998) demonstrates the efficacy of the technique for introducing small molecules into adherent cells.
The need for a method of quantifying the extent of gap junctions in adherent cells was highlighted in Fick et al. (1995) wherein it was noted that the microinjection technique in particular would require a prohibitively large number of experiments. Raptis et al. (2005) described how the apparatus disclosed in U.S. Pat. No. 5,232,856 may be employed to study populations of electroporated and non-electroporated cells as they grow side by side on a partially conductive microscope slide, how to observe whether cells have gap junctions, and how to determine the amount of gap junction communication that takes place. However, drawbacks to that technique include detachment of adherent cells during placement or removal of the upper electrode, and the inability to observe the cells until the upper electrode has been removed after the electroporation event.